To allow a study of β-catenin mutations in hepatocellular carcinomas (HCCs) induced by exogenous and endogenous carcinogens, we induced tumors in male Fischer 344 rats with N-nitrosodiethylamine and a choline-deficient l-amino acid-defined diet. Administration of the former was followed by partial hepatectomy with colchicine to induce cell cycle disturbance and a selection pressure regimen (K. Ohashi et al., Cancer Res., 56: 3474–3479, 1996; M. Tsutsumi et al., Jpn. J. Cancer Res., 87: 5–9, 1996). HCCs were obtained after 42 weeks. With continuous choline-deficient l-amino acid-defined feeding, tumors were sampled after 75 weeks. Total RNA was extracted from individual lesions and mutations in the glycogen synthase kinase-3β phosphorylation consensus motif of β-catenin were investigated by reverse transcriptase-PCR-single-strand conformation polymorphism analysis followed by nucleotide sequencing. Changes were detected in 5 of 11 HCCs induced by the exogenous carcinogen. The observed shifts of C:G→G:C or C:G→A:T at codon 33 and G:C→T:A transversions at codon 34 were associated with β-catenin protein accumulation and confirmed by Western blot analysis. Only 2 of 15 HCCs induced in the endogenous carcinogenesis regimen demonstrated mutations, those being transitions of C:G→T:A at codon 41 without amino acid alteration. These results suggest that different genetic pathways underlie exogenous and endogenous liver carcinogenesis in rats.
Liver carcinogenesis can be divided into two categories: carcinogenesis induced by exogenous carcinogen changes and that induced by endogenous changes that occur without any established carcinogen exposure. DEN3 is one of the most well-known liver carcinogens in rats. We have reported that a cell cycle disturbance induced in DEN-initiated hepatocytes by colchicine gives a growth advantage to putative preneoplastic lesions under conditions of partial hepatectomy and selection pressure, so that a high incidence of HCCs can be obtained within a short latent period (1, 2). We have also demonstrated high yields of HCCs associated with cirrhosis caused by chronic administration of a CDAA diet that does not contain any known carcinogens (3). Because our studies revealed differential effects of chemopreventive agents in our two liver models (4), there is a possibility that different mechanisms underlie exogenous and endogenous hepatocarcinogenesis in rats.
The β-catenin protein, a submembranous component of the cadherin-mediated cell-cell adhesion system, has been demonstrated to be a downstream activator of Wnt signal transduction (5, 6). The amounts of cytoplasmic β-catenin are mainly regulated by interaction with APC protein and by phosphorylation at serine and/or threonine residues through the action of GSK-3β (7). Mutations of APC or β-catenin affect the degradation of β-catenin protein by the ubiquitin/proteasome system and result in its stabilization and accumulation within cells. Accumulated β-catenin may translocate into the nucleus, where it interacts with transcription factors of the T-cell factor/lymphoid enhancer factor family to activate target genes. It has been suggested that the Wnt-β-catenin pathway may play an important role in the development of cancers (8, 9), and recent reports have documented the existence of β-catenin mutations in HCCs of human (10, 11) and the mouse (11). In this study, rat HCCs due to exogenous and endogenous carcinogens were investigated for β-catenin alterations to assess whether differences may exist in this respect.
Materials and Methods
Male Fischer 344 rats, 5 weeks old, were purchased from Japan SLC Inc. (Shizuoka, Japan) and housed in stainless steel, wire-bottomed cages in an air-conditioned room with a constant temperature of 25°C and a 12-h light-dark cycle. Food and water were given ad libitum throughout the study. After a 1-week acclimation period on a basal diet in pellet form (Oriental MF Diet; Oriental Yeast Co., Ltd., Tokyo, Japan), the animals were allocated to experimental groups.
Chemicals and Diets
DEN was purchased from Wako Pure Chemical Co., Ltd. (Kyoto, Japan) and diluted with a 0.9% NaCl solution to a concentration of 0.1%. Colchicine was purchased from Sigma Chemical Co. (St. Louis, MO) and dissolved in a 0.9% NaCl solution to a concentration of 0.05%. AAF and carbon tetrachloride (CCl4) were purchased from Nacalai Tesque, Inc. (Kyoto, Japan), and the latter was diluted 1:1 with corn oil. The diet containing 0.02% AAF was prepared by admixing the chemical with Oriental MF powdered basal diet. The CDAA diet, with the composition described previously (3), was purchased from Dyets Inc. (Bethlehem, PA) and stored at 4°C immediately upon arrival.
The method for the production of HCCs was as described previously (1, 2). Animals received DEN i.p. at a dose of 10 mg/kg body weight, followed after 4 h by partial hepatectomy performed by the method described by Higgins and Anderson (12). Colchicine at a dose of 0.5 mg/kg body weight was injected i.p. 1 and 3 days after DEN treatment. After an 11-day recovery period, rats were placed on the selection regimen, comprising feeding of 0.02% AAF diet for 2 weeks and a single intragastric administration of CCl4 at 1 ml/kg body weight, following the procedure described by Cayama et al. (13), and were killed under ether anesthesia 42 weeks after the beginning of the experiment.
Animals were continuously given the CDAA diet and killed under ether anesthesia 75 weeks after the beginning of the experiment.
At sacrifice, the livers were immediately excised, and grossly apparent tumors were dissected from surrounding tissue. Samples were frozen in liquid nitrogen and stored at −80°C until analysis. Portions of the tumors were also fixed in 10% formalin for routine processing and staining with H&E for histological examination.
Reverse Transcriptase-PCR-SSCP Analysis of the β-Catenin Gene
Total RNA was extracted from frozen tissue using Isogen (Nippon Gene, Inc., Toyama, Japan) and first-strand cDNA was synthesized from 2-μg samples with Ready-To-Go Your-Prime First-Strand Beads (Pharmacia Co. Ltd., Tokyo, Japan). To eliminate possible false-positives caused by residual genomic DNA, we treated all samples with DNase. The appropriate oligonucleotide primers were 5′-GCTGACCTGATGGAGTTGGA-3′ (sense) and 5′-GCTACTTGCTCTTGCGTGAA-3′ (antisense). Using this primer set, we could analyze the consensus sequence for GSK-3β phosphorylation of the β-catenin gene, as described previously (14). PCR for SSCP was performed in 10 μl of reaction mixture consisting of 1 μm each primer, 200 μm each dNTP, 1× PCR buffer (Perkin-Elmer, Applied Biosystems Division, Foster City, CA), 68 nm [α-32P]dCTP, 2.5 units of AmpliTaq (Perkin-Elmer), and 0.5 μl of synthesized cDNA mixture. PCR amplification was performed under the following reaction conditions: a denaturation step for 5 min at 95°C; 35 cycles of 1 min at 95°C, 1 min at 60°C, and 2 min at 72°C; and a final extension for 10 min at 72°C. PCR products were diluted with 90 μl of loading solution containing 90% formide, 20 mm EDTA, and 0.05% xylene cyanol and bromphenol blue; denatured at 90°C for 2 min; and applied to 5% polyacrylamide gels containing 0.5× Tris-borate EDTA buffer, with or without 10% glycerol. Electorophoresis was performed at 40 W for ∼2.5 h at 20°C. Gels were dried on filter paper and used to expose X-ray films at −80°C.
Cloning and Sequence Analysis
DNA fragments of mobility-shifted bands by SSCP analysis were extracted from the gels and reamplified. The PCR products obtained were cloned using a TOPO TA cloning kit (Invitrogen, San Diego, CA), and recombinant plasmid DNA clones were sequenced by Sequencing Pro (Toyobo Co. Ltd., Tokyo, Japan).
Western Blot Analysis for β-Catenin
Proteins were extracted from HCCs and Western blot analysis was carried out as described previously (14). Briefly, aliquots of 40 μg of total protein were electrophoresed on 10% SDS-polyacrylamide gels, transferred to membranes, and probed with monoclonal mouse anti-β-catenin antibody (Transduction Laboratories, Lexington, KY). The membranes were developed with a chemiluminescence system (ECL detection reagents; Amersham, Buckinghamshire, United Kingdom).
Results and Discussion
The 11 HCCs induced by DEN in 11 rats and 15 HCCs induced by the CDAA diet in 15 rats used for the analysis were all histologically well-differentiated.
The results of SSCP analysis of β-catenin gene mutations in the first group are shown in Fig. 1,A. Fragments showing abnormal mobilities, indicative of gene mutation, were observed in 5 of 11 HCCs. These results were further confirmed by nucleotide sequencing (Fig. 2). Of the five mutations in the HCCs, two were transversions at codon 33 [TCT→TGT (Ser→Cys) and TCT→TAT (Ser→Tyr)], and three were transversions at codon 34 [GGA→GTA (Gly→Val)]. It has been considered that the serine and threonine sites located in codons 33, 37, 41, and 47 in β-catenin are important for GSK-3β phosphorylation, and codons 32 and 34, which neighbor a serine, are also supposedly necessary for the ubiquitin-dependent proteolysis system (8, 9, 10, 11, 14). Missense mutations at these sites appear to be important for accumulation of β-catenin protein in cells (8, 9, 10, 11, 14). Using Western blot analysis, we confirmed such accumulation in HCCs with mutations, compared with normal liver (Fig. 3). Two of 15 HCCs induced by the CDAA diet showed abnormal mobilities by SSCP analysis (Fig. 1,B), both with ACC→ACT (Thr→Thr) transitions at codon 41, without amino acid alteration (Fig. 2). No accumulation of β-catenin protein was found in HCCs caused by DEN and the CDAA diet without missense mutations (data not shown).
The findings for mutations detected within specific codons of β-catenin and the corresponding amino acid substitutions in the protein are summarized in Table 1. Previously, it has been reported that DEN induces G→A transitions, resulting in major base substitutions (15). However, in this study, β-catenin mutations in HCCs induced by the DEN were only C→G, C→A, and G→T transversions, although G and C were target base residues for point mutations. Thus, these transversions may have been due not to DEN per se but to some other factors during liver carcinogenesis, perhaps after initiation. Similarly, the relative lack of β-catenin mutations in HCCs induced by the CDAA diet were not in line with expectation. We previously reported that 8-hydroxyguanine, a representative feature of oxidative DNA damage, is generated by the CDAA diet (4), and it is well-established that 8-hydroxyguanine induces G→T or A→C transversions in Escherichia coli (16).
It is accepted that multiple genetic alterations are necessary for the development of cancer. There have been several reports of gene mutations in HCCs induced in exogenous and endogenous carcinogenesis models. p53 and Ki-ras gene mutations were found to be absent or low frequent in rat HCCs induced by a nitrosamine (17). Although a high rate of p53 mutations was detected in rat HCCs induced by feeding a choline-deficient diet (18), we previously reported no p53 mutations and only a low frequency of Ki-ras mutations in tumors induced by the CDAA diet (19). Because β-catenin mutations in HCCs induced by DEN were relatively frequent in this study, such alterations might play an important role in their development. This appears less likely to be the case for our endogenous hepatocarcinogenesis. Recently, we described hypomethylation of the c-myc gene in HCCs induced by the CDAA diet in rats, suggesting that altered DNA methylation might be of essential significance in this model.4
In conclusion, we have demonstrated different frequencies and patterns of β-catenin mutations in rat HCCs induced in exogenous and endogenous carcinogenesis models. The results suggest that different genetic pathways may be involved in the two cases, which has possible implications for chemoprevention and therapeutic approaches.
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This work was supported in part by Grants-in Aid for Cancer Research 7-1 and 8-2 (to Y. K.) and 10-4 (to T. T.) from the Ministry of Health and Welfare of Japan; Grants-in Aid for Scientific Research Expenses for Health and Welfare Programs, 2nd-Term Comprehensive 10-Year Strategy for Cancer Control, Cancer Prevention, from the Ministry of Health and Welfare of Japan (to Y. K.); and Grant-in-Aid 08264108 for Scientific Research in Priority Areas, Cancer Research, from the Ministry of Education, Science, Sports and Culture of Japan (to Y. K.).
The abbreviations used are: DEN, N-nitrosodiethylamine; HCC, hepatocellular carcinoma; CDAA, choline-deficient l-amino acid-defined; GSK-3β, glycogen synthase kinase 3β; AAF, 2-acetylaminofluorene; SSCP, single-strand conformation polymorphism.
T. Tsujiuchi, M. Tsutsumi, Y. Sasaki, M. Takahama, and Y. Konishi. Hypomethylation of CpG sites and c-myc gene overexpression in HCCs but not hyperplastic nodules induced by a choline-deficient l-amino acid-defined diet in rats, submitted for publication.
|Sample no. .||Mutated codon no. .||Base change .||Amino acid substitution .|
|Sample no. .||Mutated codon no. .||Base change .||Amino acid substitution .|
We thank Rie Maeda and Yumi Horikawa for their assistance with the preparation of this manuscript.